A putative SUMO interacting motif in the B30.2/SPRY domain of rhesus macaque TRIM5α important for NF-κB/AP-1 signaling and HIV-1 restriction

Abstract

TRIM5α from the rhesus macaque (TRIM5αRh) is a restriction factor that shows strong activity against HIV-1. TRIM5αRh binds specifically to HIV-1 capsid (CA) through its B30.2/PRYSPRY domain shortly after entry of the virus into the cytoplasm. Recently, three putative SUMO interacting motifs (SIMs) have been identified in the PRYSPRY domain of human and macaque TRIM5α. However, structural modeling of this domain suggested that two of them were buried in the hydrophobic core of the protein, implying that interaction with SUMO was implausible, while the third one was not relevant to restriction. In light of these results, we re-analyzed the TRIM5αRh PRYSPRY sequence and identified an additional putative SIM (435VIIC438) which we named SIM4. This motif is exposed at the surface of the PRYSPRY domain, allowing potential interactions with SUMO or SUMOylated proteins. Introducing a double mutation in SIM4 (V435K, I436K) did not alter stability, unlike mutations in SIM1. SIM4-mutated TRIM5αRh failed to bind HIV-1CA and lost the ability to restrict this virus. Accordingly, SIM4 undergoes significant variation among primates and substituting this motif with naturally occurring SIM4 variants affected HIV-1 restriction by TRIM5αRh, suggesting a direct role in capsid recognition. Interestingly, SIM4-mutated TRIM5αRh also failed to activate NF-κB and AP-1-mediated transcription. Although there is no direct evidence that SIM4 is involved in direct interaction with SUMO or a SUMOylated protein, mutating this motif strongly reduced co-localization of TRIM5αRh with SUMO-1 and with PML, a SUMOylated nuclear protein. In conclusion, this new putative SIM is crucial for both direct interaction with incoming capsids and for NF-κB/AP-1 signaling. We speculate that the latter function is mediated by interactions of SIM4 with a SUMOylated protein involved in the NF-κB/AP-1 signaling pathways.

RanBP2 regulates the anti-retroviral activity of TRIM5α by SUMOylation at a predicted phosphorylated SUMOylation motif

“…Although TRIM5α forms cytoplasmic bodies, it can also shuttle to the nucleus 12 , 13 , suggesting that it could interact with SUMO machinery there. Some have shown co-localization of overexpressed TRIM5α with Promyelocytic Leukaemia Nuclear Bodies (PML-NB) 13 , 14 , which are a preferred site of SUMOylation and harbor many proteins of the SUMO machinery, including SUMO and PIAS1 15 , 16 , however we could not reproduce these findings with endogenous TRIM5α 13 . Therefore, although PIAS1 can SUMO modify TRIM5α in vitro 11 , their distinct endogenous localizations in cells suggests that PIAS1 is unlikely to act as the main E3 SUMO ligase of TRIM5α in vivo.…”

“…Besides direct conjugation of TRIM5α by SUMO variants 11 , which we confirm in this study, the SUMO machinery is likely to contribute in additional ways to TRIM5α function. Several SUMO-interacting motifs (SIM) identified in TRIM5α 9 , 10 , 14 may mediate interaction with SUMOylated proteins. For instance, the physical association between TRIM5α and RanGAP1 that we describe in this study likely involves an interaction between SUMOylated RanGAP1 and SIM domains on TRIM5α, and may be key to bring TRIM5α in proximity of its E3 SUMO ligase, RanBP2.…”

HIV-1 capsids from B27/B57+ elite controllers escape Mx2 but are targeted by TRIM5α, leading to the induction of an antiviral state

“…The TRIM5α N-terminal RING domain recruits the E2-ubiquitin conjugating enzyme heterodimer Ubc13 (Ube2N)/Uev1a (or Uev2) to generate lysine 63 (K63)-linked polyubiquitin chains [ 34 , 35 ] that can be anchored onto TRIM5α through the action of another E2 enzyme, Ube2W [ 32 , 33 ]. K63-linked ubiquitin, in association with the TAK1 kinase complex, leads to the activation of both NF-κB and AP-1 pro-inflammatory pathways [ 33 , 34 , 36 , 37 ].…”

Hierarchical assembly governs TRIM5α recognition of HIV-1 and retroviral capsids

“…3A). The V435K/I436K mutations within this segment were previously shown to disrupt capsid binding and restriction activity ( 24 ).…”